Tumor Necrosis Factor, or more specifically Tumor Necrosis Factor-alpha (TNF-.alpha.), is a cytokine, primarily produced by stimulated macrophages, that exhibits not only a striking cytotoxicity against various rumour cells [Carswell et al., Proc. Nat. Acad. Sci., U.S.A. 72, 3666-3670, (1975)] but also plays a multiple role as a mediator of inflammation and the immune response [for an overview see Beutler and Cerami, Ann. Rev. Immunol. 7, 625-655 (1989); Bonavista and Granger (eds.) "Tumor Necrosis Factor: Structure, Mechanism of Action, Role in Disease and Therapy, Karger, Basel (1990)]. The primary structure of human Tumor Necrosis Factor-alpha (hTNF-.alpha.) has been deduced from the nucleotide sequence of a eDNA which has been cloned and expressed in E. coli [Pennica et al., Nature 312, 724-729 (1984); Marmenout et al., Eur. J. Biochem. 152, 515-522 (1985); Wang et al., Science 228, 149-154 (1985); Shirai et al., Nature 313, 803-806 (1985)]. A striking homology in amino acid sequence (30%) was found between hTNF-.alpha. and human Lymphotoxin, often referred to as human Tumor Necrosis Factor-beta (hTNF-.beta.), a cytokine mainly produced by lymphocytes [Gray et al., Nature 312, 721-724 (1984); Fiers et al., Cold Spring Harbour Symp. 51, 587-595 (1986)].
h TNF-.alpha. with modified amino acid sequences, so called TNF-.alpha.-muteins, have also been described in the art [for example see Yamagishi et al., Protein Engineering 3, 713-719, (1990) or Fiers in "Tumor Necrosis Factors: Structure, Function and Mechanism of Action", Aggarwal and Vilcek (eds.), Marcel Dekker, Inc., New York, in press, or Fiers et al. in Bonavista and Granger, pp. 77-81 (s.a.)]. In addition TNF-.alpha.-muteins have also been the object of several patent applications, e.g. International Patent Applications Publ. Nos. WO 86/02381, WO 86/04606, WO 88/06625 and European Patent Applications Publ. Nos. 155,549; 158,286; 168,214; 251,037 and 340,333, and Deutsche Offenlegungsschrift Nr. 3843534.
Muteins of Lymphotoxin have also been disclosed in the art, e.g. in European Patent Applications Publ. Nos. 250,000; 314,094 and 336,383.
The biological effects of TNF are mediated via specific receptors, namely a receptor with an apparent molecular weight of 55 kD on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (p55-TNF-R) and a receptor with an apparent molecular weight of 75 kD on SDS-PAGE (p75-TNF-R). Both forms of TNF-receptor have been cloned, p55-TNF-R by Loetscher et al. [Cell 61, 351-359, (1990)] and p75-TNF-R by Dembic et al. [Cytokine 2, 53-58, (1990)] (for both receptors see also European Patent Application No. 90116707.2). It was recently found that both receptors bind not only TNF-.alpha. but also TNF-.beta. with high affinity [Schonfeld et al., J. Biol. Chem. 266, 3863-3869 (1991)].
It is well known in the art that on the basis of its biological activities TNF-.alpha. can be a valuable compound for the treatment of various disorders. For example TNF-.alpha., alone or in combination with interferon, can be an effective antitumor agent [Brouckaert et al., Int. J. Cancer 38, 763-769 (1986)]. However, its systemic toxicity is a limit to its wider therapeutic use [Taguchi T. and Sohmura Y., Biotherapy 3, 177-186 (1991)].
It has been shown that human TNF-.alpha. (hTNF-.alpha.) in mice only binds to the 55 kD mouse TNF receptor (murine p55-TNF-R) and is far less toxic than murine TNF-.alpha. (mTNF-.alpha.), which binds to both p55-TNF-R and p75-TNF-R. In C57B16 mice, the LD50 of mTNF.alpha. is about 10 .mu.g/mouse and the LD50 of hTNF-.alpha. is about 500 .mu.g/mouse [Brouckaert et al., Agents and Actions 26, 196-198 (1989); Everaerdt, B. et al., Biochem. Biophys. Res. Comm. 163, 378-385 (1989); Lewis, M. et al., Proc. Natl. Acad. Sci. USA 88, 2830 (1991)]. Hence the p75-TNF-R seems to play a special role in systemic toxicity.
hTNF-.alpha. and mTNF-.alpha. bind almost equally well to human p55-TNF-R (hp55-TNF-R) and to human p75-TNF-R (hp75-TNF-R). However, hTNF-.alpha. mutants, which have retained the biological activity mediated by hp55-TNF-R but have lost nearly all activity mediated by hp75-TNF-R, are the functional equivalent of hTNF-.alpha. in the murine system in that they are expected to have reduced systemic toxicity in primates just as hTNF-.alpha.. has reduced toxicity in mice and fails to bind mouse p75-TNF-R.
Human Tumor Necrosis Factor muteins showing a significant difference between their binding affinity to the human p75-Tumor-Necrosis-Factor-Receptor (hp75-TNF-R) and to the human p55-Tumor-Necrosis-Factor-Receptor (hp55-TNF-R), have been described in European Patent No. 486 908.